Outward-oriented sites within clustered CTCF boundaries are key for intra-TAD chromatin interactions and gene regulation

CTCF plays an important role in 3D genome organization by adjusting the strength of chromatin insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in a tandem array with a complex divergent or convergent orientation. Here, using Pcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in mice in vivo or CBS elements in cultured cells in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intra-TAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries in developmental gene regulation and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection No software was used for data collection

Data analysis
For 4C data, Duplicated paired-end reads were removed by FastUniq (version 1.1) program, and only the unique reads were used for analyses using the Bowtie and r3Cseq program.Paired-end reads were aligned to the mouse genome (M.musculus, UCSC mm9) or the human genome (H.sapiens, UCSC hg19) using Bowtie2(version 2.3.5).The sam files were then transformed into bam file using samtools (version 1.15.1).The reads per million (RPM) value was calculated using the r3Cseq program (version 1.20) in the R package (version 3.3.3).For ChIP-seq and ATAC-seq data, Reads were aligned to the mouse genome (M.musculus, UCSC mm9) or the human genome (H.sapiens, UCSC hg19) with modification in the CRISPR editing area if necessary using Bowtie2(version 2.3.5).Sam files were then converted into Bam files using samtools (version 1.15.1) and indexed.BedGraph files were generated using bigWigToBedGraph software with Bam file as input.Bw files were generated using macs14 (version 1.4) software with Bam files as input.For RNA-seq data, Reads were aligned using Hisat2 (version 2.0.4) to the human genome (GRCh38/hg38), The sam files were then transformed into bam file using samtools (version 1.15.1) and the FPKM value was calculated using the Cufflinks program (version2.1.1).Hi-C reads were pre-processed with HiC-Pro (version 3.0.0).
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.Abcam ab992 For each experiment,2 ul of antibodies were added with no dilution.H3K27ac Abcam ab4729 For each experiment,3 ul of antibodies were added with no dilution.H3K4me1 Abcam ab8895 For each experiment,3 ul of antibodies were added with no dilution.H3K4me3 Millipore 17-614 For each experiment,3 ul of antibodies were added with no dilution.H3K9ac Millipore 06-942 For each experiment,3 ul of antibodies were added with no dilution.

H3K36me3
Abcam ab9050 For each experiment,3 ul of antibodies were added with no dilution.H3K9me2 Abcam ab1220 For each experiment,3 ul of antibodies were added with no dilution.H3K9me3 Millipore 07-442 For each experiment,3 ul of antibodies were added with no dilution.

Validation
For CTCF antibody, the company is Millipore with cat.Number 07-729.It is a polyclonal antibody.According to the Millipore website, They routinely evaluate the antibody by Western Blot (Western Blot Analysis: A 1:1000-1:5000 dilution of this lot detected CTCF in HeLa nuclear extract.A previous lot detected CTCF in K562 nuclear extract).For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.For Rad21 antibody, the company is Abcam with cat.Number ab992.It is a polyclonal antibody.According to the Abcam website, they guarantee the antibody has been tested for IP and WB.For each ChIP-seq experiment, 2 ul of antibodies were added with no dilutions.For H3K27ac antibody, the company is Abcam with cat.Number ab4729.It is a polyclonal antibody.According to the Abcam website, they guarantee the antibody has been tested for ICC/IF, WB, IHC-P, ChIP and Preparr.For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.For H3K4me1 antibody, the company is Abcam with cat.Number ab8895.It is a polyclonal antibody.According to the Abcam website, they guarantee the antibody has been tested for ICC/IF, ChIP, WB and IHC-P.For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.For H3K4me3 antibody, the company is Millipore with cat.Number 17-614.It is a monoclonal antibody ( clone name unprovided according to Merck).According to the Millipore website, they routinely evaluate the antibody by Chromatin Immunoprecipitation (Sonicated chromatin prepared from untreated HeLa cells (1 X 106 cell equivalents) was subjected to chromatin immunoprecipitation using 3 μL of either a normal rabbit IgG or 3 μL Anti-Trimethyl-Histone H3 (Lys4) Monoclonal IgG and the Magna ChIP A (Part # 17-610) Kit.Successful immunoprecipitation of trimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using control ChIP Primers flanking the human GAPDH promoter).For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.For H3K9ac antibody, the company is Millipore with cat.Number 06-942.It is a polyclonal antibody.According to the Millipore website, they routinely evaluate the antibody by Western Blotting Analysis (A 1:10,000 dilution of this antibody detected acetyl-Histone H3 (Lys9) in 10 μg of sodium butyrate treated HeLa cell lysate).For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.For H3K36me3 antibody, the company is Abcam with cat.Number ab9050.It is a polyclonal antibody.According to the Abcam website, they guarantee the antibody has been tested for ICC/IF, WB and ChIP.For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.For H3K9me2 antibody, the company is Abcam with cat.Number ab1220.It is a monoclonal antibody with clone number mAbcam 1220.According to the Abcam website, they guarantee the antibody has been tested for ICC/IF, WB, ELISA, IHC-P and ChIP.For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.For H3K9me3 antibody, the company is Millipore with cat.Number 07-442.It is a polyclonal antibody.According to the Millipore website, they routinely evaluate the antibody by Western Blot on HeLa acid extract and recombinant Histone H3 (cat.# 14-411) (Western Blot Analysis: 1:500 dilution of this lot detected trimethyl Histone H3 on 10 μg of HeLa acid extract but not on recombinant Histone H3).For each ChIP-seq experiment, 3 ul of antibodies were added with no dilutions.

Eukaryotic cell lines
-729 For each experiment,3 ul of antibodies were added with no dilution.Rad21 Policy information about cell lines and Sex and Gender in ResearchCell line source(s)